Real-Time Fluorescence Quantitative PCR Analysis Of Bacterial Microfloras In Chronic Venous Leg Ulcers

Objective:The bacterial microfloras in chronic venous leg ulcers play a important role in the healing process, but there is still considerable debate as to the importance of individual species or microbial density in relation to healing.In our study,we analyzed the relationship between the bacterial groups frequently recovered from chronic venous leg ulcers and ulcer healing.Methods:All patients with chronic venous leg ulcers included in this study were treated with standard regimen of compression therapy,Patients were followed up for 6 months to determine healing rates of ulcers,Ulcers with 100% closure of the wound and did not reoccur within 1 month of wound closure were included in the healing group.Tissue samples from ulcer bed were obtained by biopsy prior to treatment,DNA was extracted directly from the tissue sample. A set of universal primer and group or species-specific primers for the bacterial groups(Staphylococcus aureus,Pseudomonas aeruginosa,Staphylococcus epidermidis, Enterococcus,Peptostreptococcus,Bacteroides) frequently recovered from CVLU were synthesized.The bacterial difference between healing and nonhealing chronic venous leg ulcers was compared using PCR and real-time fluorescence quantitative PCR.Resluts:The Bacteria were detected in all ulcers.In healing and nonhealing groups,the most common bacteria were Staphylococcus aureus (67.9% and 57.1%,respectively),Pseudomonas aeruginosa (35.7% and 42.9%,respectively). Quantitative polymerase chain reaction was used to compare the total bacterial load and the density of Staphylococcus aureus and Pseudomonas aeruginosa at two different groups,The total bacterial load in nonhealing chronic venous leg ulcers(6.38±0. 78vs 5. 39±0. 99) significantly higher than those of healing ( P < 0. 05) , whereas the density of Staphylococcus aureus (2. 71±0. 66vs 2. 82±1.07) and Pseudomonas aeruginosa ( 2. 42±0.42 vs 2. 51±0. 70) did not show significant difference ( P >0. 05).Conclusion : In our study,no single bacterial species or group frequently recovered from CVLU was shown to be associated with healing outcome. A significant association between healing and the total bacterial load in the wound was demonstrated.This study suggests that the increase of total bacterial load and Bacterial biofilms play important roles in maintaining a chronic inflammation state that ultimately leads to the failure of chronic venous leg ulcers to heal.